I am using Agilent 1200 with openLAB CDS for the analysis that I am doing. I am plotting a standard calibration curve but I observed that the peak area does not increase as the concentration increases. The peak area provided by the integration output does not follow a defined order. Does anyone know how to solve this problem? I hope to hear from someone soon.

Hello kingsley, welcome! Which OpenLAB are you using (choices are ChemStation Edition, EZChrom Edition, or simply CDS 2.0, 2.1, or 2.2) and what revision? You can find out both of these pieces of information if you click Help > About; both the type of software and Revision should be right in that window.

I am assuming you’re using ChemStation Revision C.01.06 in that case. Can you confirm that’s true? I should clarify that Help > About in the actual analytical software should reveal the correct information we’re looking for. In other words, don’t look for the Revision in the OpenLAB Control Panel.

Sorry to harp on this, but the advice you get will vary significantly based on the software type and Revision.

Ok, thanks for confirming! So, let’s tease out what you’re trying to accomplish. When you say that the areas are not increasing with increasing concentration, which part of the software are you referring to? If you’re looking at the calibration table, it sounds to me like perhaps you haven’t loaded more than one data file when building your concentration levels. Since by your question you’re implying that you are trying to build a multi-level calibration curve, you should have a separate corresponding data file for each concentration level. When building the curve, load the data file corresponding to the first concentration, create the first level, then move to the next file and so on.

So you’re saying that when you inject increasingly higher concentrations for example, the data files you obtain in ChemStation don’t show higher peak areas? In that case, I would guess that either the sample preparation is problematic or the concentrations you’re injecting are outside the linear dynamic range of the detector. It might help to post your method parameters and the chromatograms that show the discrepancy to which you’re referring.

Yes that is the problem. I am using porosell 120 C18 column with internal diameter of 4.6 and length of 50mm. The mobile phase is 0.1% Formic acid + water (A) and 0.1% Formic acid + Acetonitrle (B). the gradient elution time table is stated is as follows: 90% A and changing to 90% B at 6mins and finally returing to the initial conditions at 12mins.

The calibration standards are prepared from 196 ul of potassium phosphate buffer at pH 7.4 and 5uL of microsomal protein followed by the addition of 1uL of standard working solutions(2ug/ml,3ug/ml,24ug/ml,48ug/ml,180ug/ml and 420ug/ml) and 10ul of acetonitrile containing internal standard was added to reach the final concentration. The solution was centrifuged and filtered using a centrifugal filter device and 10uL of the filtrate was injected onto the LC.

This method is been validated for the drug metabolism studies that am doing.

Hi kingsley, did you get a chance to find some representative chromatograms? As I said in a prior post, either the sample preparation is problematic or the concentrations being objected are outside the quantitation range. This does not appear to be a problem with the software, but there’s insufficient information to draw a conclusion. Are you using internal standard quantitation?

Do you still need any help with this issue or have you found a working solution? Please let us know if you need any further assistance and we can pick it up from there. If you have resolved the problem, it would be appreciated if you could reply to this post with the solution and mark it as the "Correct Answer".

If you still need help with this, then I recommend that you follow up with valentinrusu's last reply. There are some questions posed in that reply that will need to be answered in order to move forward with troubleshooting.